Regulation of polypeptide chain initiation in Chinese hamster ovary ...

Val. 262, No. 2, Issue of January 15,pp. 767-771 1987 Printed in d.S.4.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Regulation of Polypeptide Chain Initiation in Chinese Hamster Ovary Cells with a Temperature-sensitive Leucyl-tRNA Synthetase CHANGES IN PHOSPHORYLATION OF INITIATION FACTOR eIF-2 AND IN THE ACTIVITY OF THE GUANINE NUCLEOTIDEEXCHANGE FACTOR GEF* (Received for publication, June 23, 1986)

Michael J. ClemensSQ, Angela Galpine*, Sara A. Austin$, Richard Panniersll, Edgar C. Henshawl, Roger Duncan11, J o h n W. B. Hershey 11, and Jeffrey W. Pollard** From the $Cancer Research Campaign Mammalian Protein Synthesis and Interferon Research Group, Department of Biochemistry, St. George's Hospital Medical School, Cranmer Terrace, London S W17 ORE, United Kingdom, the Warner Center, Uniuersity of Rochester Medical Center, Rochester, New York 14642, the 1) Department of Biological Chemktty, School of Medicine, University of California, Davis, California 95616, and the **Medical Research Council Group in Human Genetic Disease, Department of Biochemistry, King's College, Campden Hill, London W8 7AH, United Kingdom

When cultures ofthe temperature-sensitive Chinese shown that the rapid inhibition of initiation which develops hamster ovarycell mutant tsHl are shifted from34 "C is due to a defect in the ability of initiation factor eIF-2 to (permissive temperature) to 39.5 OC (nonpermissive form 40 S ribosomeSMet-tRNAf initiation complexes (6, 7). temperature), protein synthesis is inhibited by more In extracts from amino acid-starved cells, addition of eIF-2' t h a n 80%. This is due principallyto a block in activity was able to reverse the inhibition of protein synthesis (7). of polypeptide chain initiation factor eIF-2. I n t h i s By using a series of mutants of Chinese hamster ovary cells paper we show that there is impairment of the ability with temperature-sensitive lesions in individual aminoacylof the guanine nucleotide exchange factor (GEF) to tRNA synthetases, it has been possible to demonstrate that, displace GDP from eIF-2.GDP complexes inextracts at the nonpermissive temperature,these cells behave like from cells incubated at the nonpermissive temperature. amino acid-starved cells and exhibitdefective protein syntheAddition of GEF or of high concentrations of eIF-2 sis at thelevel of eIF-2 activity and polypeptide chain initiastimulates protein synthesis to the level observed in control cell extracts, suggesting that GEF is rate-lim- tion (9). This behavior was not observed in the corresponding iting for eIF-2 activity a n d overall protein synthesis wild-type CHO cells. These observations suggested that the at the nonpermissive temperature. Analysis of eIF-2 effects of amino acid starvation maybe exerted through by two-dimensional gel electrophoresis and immuno- changes in aminoacyl-tRNA synthetase activity and that a regulatory relationship must exist between these enzymes and blotting reveals an increase i n the proportion of the subunit inthe phosphorylated form from5.5 f 2.4% to initiation factor eIF-2. However, it was not clear as to how 17.2 f 3.9%on shifting tsH, cells from 34 to 39.5 "C. eIF-2 activity and overall initiation could be controlled in No such effect is seen in wild-type cells, which do not such a system. In particular, it was of interest to determine exhibit temperature-sensitive protein synthetic activ- whether the mechanisms of translational control observed in ity. Since the primary lesion in tsHl cells is in their heme-deficient reticulocyte lysates, viz. partial phosphorylaleucyl-tRNA synthetase, these results suggest a role tion of the a subunit of eIF-2 (10, 11) and consequent inhifor eIF-2 phosphorylation and GEF activity in coupling bition of activity of the guanine nucleotide exchange factor the r a t e of polypeptide chain initiation to the activity GEF (also called eIF-2B), were operative in this nonerythroid of thechain elongation machinery. cell system. In the present work, we have used the temperature-sensitive leucyl-tRNA synthetase mutant of CHO cells, tsH, (161, to demonstrate that, as in heme-deficient reticulocytes, inhibition of initiation is associated with a substantial In mammalian cells, the overall rate of protein synthesis is impairment of GEF activity and that protein synthesis in regulated at thetranslational level bymany different changes vitro can be rescued by addition of purified GEF. A small but in the external environment. Well documented examples in- statistically significant increase in the phosphorylation of the clude the effects of heat shock (1, 2), availability of serum or a subunit of eIF-2 accompanies the inhibition of GEF activity. growth factors (3, 4), and the supply of essential nutrients such as glucose (5) or amino acids (5-8). In all cases control EXPERIMENTALPROCEDURES is exerted primarily at thelevel of polypeptide chain initiation Materials-~-[~S]Methioninewas purchased from New England and, in thecase of amino acid starvation, we have previously (Y

* This work was supported by a Career Development Award and

grant from the Cancer Research Campaign, United Kingdom (to M. J. C.), by United States Public Health Service Grants CA-21663 and CA-11198 ( t o E. C. H.), by Grant GM 22135 from the National and by a grant from the Medical Institutes of Health (to J. W. B. H.), Research Council, United Kingdom (to J. W. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement'' in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. 8 To whom correspondence should be addressed.

Nuclear and [3H]GDP from Amersham International. Acrylamide and bisacrylamide were obtained from Serra (Garden City Park, NY). Ampholytes were purchased from LKB Instruments Inc., Rockville,

MD. Cell Culture, Measurement of Protein Synthesis, and Preparation procedures have been described in detail previously (7, 9, 17). Briefly, CHO cells were grown at 34 "C (&HI) or 37 "C (wild-type) in monolayer or suspension culture in a-miniof Cell Extracts-These

The abbreviations used are: eIF, eukaryotic initiation factor;

CHO,Chinese hamster ovary; HEPES, 4-(2-hydroxymethyl)-l-piper-

767

azineethanesulfonic acid; GEF, guanine nucleotide exchange factor.

768

Control of Temperature-sensitive Initiation in

mum essential medium (18) containing 50 pg/ml asparagine. HzOand 8%(v/v) fetal calf serum. Cells were incubated at 34 "C (permissive temperature) or rapidly shifted upto 39.5 "C (nonpermissive temperature) for 45 min. In some experiments some incubations were subsequently returned to 34 "C for a further 45 min. Protein synthesis in intact cells was monitored by the incorporation into acid-insoluble material of a mixture of [3H]threonine, [3H]isoleucine, and [3H] phenylalanine (0.33 pCi/ml each) for 15 min (9, 17). For assessment of protein synthesis and GEF activity in vitro, cell extracts were prepared by lysis with 0.25% (v/v) Nonidet P-40, and postmitochondrial supernatants were obtained by centrifugation. Cell-free protein synthesis on endogenous mRNA was assayed by incorporation of [35S]methionine(100 pCi/ml) in the presence of 25 mM HEPES, pH 7.6, 110 mM KCl, 1 mM magnesium acetate, 6 mM 2-mercaptoethanol, 0.4 mM spermidine, 4% (v/v) glycerol, 1mM ATP, 0.25 mM GTP, 5 mM creatine phosphate, and 180 pg/ml creatine kinase, as described previously (7,9). Assay of GEF Activity-Guanine nucleotide exchange factor activity intsH, cell extracts was measured by the kinetics of displacement of [3H]GDP from eIF-2. GDP complexes in the presence of 100-fold excess of unlabeled GTP, as originally described for reticulocyte lysates (19). 15 pg of purified rat liver eIF-2 which was free of GEF activity (20) was incubated with 2 p~ 13H]GDP for 10 min at 30 "C in a volume of 220 pI in the presence of 20 mM HEPES, pH 7.6, 60 mM KCl, 1 mM dithiothreitol, and 100 pg/ml creatine kinase. The complexes were stabilized by addition of magnesium acetate to a final concentration of 1 mM, and 50-pl aliquots were added to 350-pl incubations containing 150 pl ,of cell extract. Displacement of [3H] GDP was monitored by incubating this mixture at 30 'C in the presence of 20 mM HEPES, 90 mM KCl, 1 mM magnesium acetate, 0.1 mM dithiothreitol, 14 pg/ml creatine kinase, and 30 p~ GTP. Aliquots of 60 pl were removed at appropriate time intervals, diluted into 20 mM HEPES, 90 mMKC1, 1 mM magnesium acetate and immediately filtered through 24-mm cellulose nitrate discs (19). These were washed three times with this buffer and dried and the bound radioactivity was determined by liquid scintillation counting. Preparation and Purification of Initiation Factors-Initiation factors eIF-2 and GEFwere prepared from mouse Ehrlich ascites tumor cells or rat liver as previously described (15, 20). To maintain its stability on storage, the GEFwas purified as a 1:l complex with eIF2. This was separated from free eIF-2 by sucrose gradient centrifugation followed by CM-Sephadex chromatography and was stored in liquid nitrogen at 300 pg/ml in 20 mM HEPES, pH7.6,100 mM KCl, 0.1 mM EDTA, 0.1 mM dithiothreitol, 10% (v/v) glycerol. Initiation factor eIF-4F was purified from rabbit reticulocytes (21) and was a generous gift from Dr. W. C. Merrick (Case Western Reserve University, Cleveland, OH). Gel Electrophoresis and Zmmumblotting-For gel analysis, cells that had been incubated at 34 or 39.5 "C were washed three times with phosphate-buffered saline and lysed as rapidly as possible in 9.8 M urea, 2% (v/v) Nonidet P-40,2% (v/v) pH 3.5-10 LKB Ampholines, 1%(v/v) 2-mercaptoethanol (approximately 400 pl/106 cells). In most experiments this buffer also contained 50 mM NaF and 10 mM EDTA to inhibit protein phosphatase and kinase activities. Debris was removed by centrifugation for 30 s in a Beckman Microfuge. The lysates were stored a t -70 "C prior to analysis. Gel electrophoresis of cell lysates in two dimensions was carried out as described previously (22). The immunoblotting techniques using affinity-purified rabbit antibodies against eIF-2a and eIF-28 or a goat antiserum against eIF-4B have also been described (1, 4, 22). Bound antibodies were detected by treating the blots with lZ5I-labeled goat anti-rabbit IgG or rabbit anti-goat IgG as appropriate, with visualization by autoradiography. The percent phosphorylation of eIF-2a was quantified by scanning the autoradiograms in two dimensions with an LKB 2202 laser densitometer linked to an Apple IIe computer. Data were analyzed using the LKB 2190 GelScan program. Approximately 15 serial scans across each pair of eIF-2a spots were performed and therelative intensity of each spotin a pair calculated from the sum of the values obtained. RESULTS

In orderto determine whether the decrease in eIF-2 activity which occurs when tsH, cells are shifted from 34 to 39.5 "C (9) is due to loss of ability of eIF-2 to recycle between successive rounds of initiation, we have examined the activity of the guanine nucleotide exchange factor GEF. This protein

Cells CHO

catalyzes the exchange of GDP on eIF-2 for GTP, a step which is essential for the reutilization of eIF-2 in protein synthesis (12-15). GEF can be conveniently assayed in cell extracts by its ability to displace [3H]GDP from added eIF2.GDP complexes in the presence of excess GTP (19). Fig. 1 shows the kinetics of GDP displacement in extracts from tsH, cells incubated at 34 and 39.5"C. Although the assays are conducted at 30 "C in all cases, extracts of cells exposed to the nonpermissive temperature show a 3-4-fold reduction in the initial rateof GDP displacement, relative to extracts from cells maintained a t 34 "C. Thus, by this criterion, GEF activity is inhibited at 39.5 "C to a similar extent aswe have observed previously for eIF-2-dependent ternary complex formation and 40 S initiation complex formation in vitro (9) and for overall protein synthesis in viuo (9, 16). To test whether GEF activity is rate-limiting for protein synthesis in extracts from cells exposed to 39.5 "C, we have added increasing amounts of a purified eIF-2. GEFcomplex to theCHO cell-free systems. For comparison, purified eIF-2 (free of GEF) was also added to the same extracts. Fig. 2 shows that both factor preparations stimulateprotein synthesis in 39.5 "C extracts, with relatively little effect on 34 "C extracts. In the experiment shown, eIF-2 completely eliminated the difference between the activities of the two extracts, and eIF-2.GEF reduced the difference to less than 20%. In some experiments we have observed greater stimulation of 34 "C extracts than shown in Fig. 2 but there was always a bigger effect on the corresponding 39.5 "C extracts (data not shown). Half-maximal stimulation of protein synthesisoccurs with protein concentrations of the eIF-2 .GEF and eIF-2 preparations of about 36 and 12.5 pg/ml, respectively. Allowing for their respective purities (30 and 80%) and molecular weights (350,000 and 125,000 daltons), these values, correspond to around 30 pmol of eIF-2. GEF/ml and 79 pmol of eIF-2/ml. These relative potencies are consistent with the possibility that the GEFis acting catalytically whereas eIF-2 alone is utilized stoichiometrically in this system. It is not clear why the eIF-2. GEFcomplex did not completely restore protein synthesis in the 39.5 "C extract, but this may have required a higher concentration than themaximum added (60 r

0

5

0 1 5 Time (mid

FIG. 1. Guaninenucleotideexchange activity incell extracts from tsHl cells incubated at 34 and 39.6 "C. Complexes were formed between purified eIF-2 and [3H]GDPas described under "Experimental Procedures." Equal amounts of these complexes were then added to extracts from tsHl cells in the presence of excess (30 p ~ unlabeled ) GTP, andincubations were carried out at30 "C under the conditions described under "Experimental Procedures." At the times indicated, 60-pl samples were removedfor determination of the eIF-2. GDP remaining by retention of the complexes on cellulose nitrate filters (19). 0,extract from cells incubated a t 34 "C;0,extract from cells incubated for 45 min at 39.5 'C. In the absence of cell extract the eIF-2. GDP complexes were completely stable for a t least 20 min a t 30 "C (data not shown).

Control of Initiation in Temperature-sensitive CHO Cells

I

I

0

w)

20

0IF-2 added (pqlrnl)

300

20 40 eIF2.GEF added (pq/ml)

60

FIG. 2. Restoration of protein syntheticactivity in extracts from tsH, cells incubated at 39.5 "C by addition of @IF-2or GEF. Cell extracts were prepared and incubated under conditions suitable for in uitro protein synthesis on endogenous mRNA. The indicated concentrations of purified eIF-2 (free of GEF) or of eIF-2. GEF complexes (both from Ehrlich ascites tumor cells) were added from zero time, and the incorporation of ["Slmethionine (100 pCi/ ml) into protein was determined in 2O-pl samples after 60 min at 30 "C. e,extract from cells incubated at 34 "C; 0, extract from cells incubated for 45 min at 39.5 "C.

TABLE I Stimulation of protein synthesis by purified initiation factorsin an extract from tsH, cells incubated at 39.5 "C The experimental conditions were as in Fig. 2, except that cell-free incubation was for only 30 min at 30 "C. Where indicated, eIF-2 (from Ehrlich cells) was added at 40 pg/ml, GEF (as a 1:l complex with eIF-2) was added at 30 pg/ml, and eIF-4F (from rabbit reticulocytes) was added at 29 udml. Factors added

Protein svnthesis cpm X IOP

2.45

GEF

eIF-2 426 eIF-2. 337 eIF-2 +GEF eIF-2. eIF-4F 107 eIF-2. GEF + eIF-4F

10.44 8.26 46711.43 2.61 682 16.70

Relative activitv %

100

~-

pg/ml). Although the eIF-2.GEF complex we have used was not completely pure, it contained no free eIF-2, having been separated from the latter by sucrose gradient centrifugation and CM-Sephadex chromatography (data not shown). It is also unlikely that some other component was responsible for the activity since the effects on protein synthesisof saturating amounts of this complex and of eIF-2 alone were similar in magnitude and not additive (Table I). The table also shows that the mRNA cap-binding factor eIF-4F, which is ratelimiting for protein synthesis after heatshock (2), on its own had no effect on the cell-free system from 39.5 "C tsHl cells. In the presence of added GEF, however, eIF-4F stimulated protein synthesisby a further factor of 2. These resultssuggest that the mRNA binding step of initiation only becomes limiting in this system in thepresence of excess active GEF (or eIF-2). In viewof the inactivation of endogenous eIF-2 (9) and GEF activity (Fig. 1)by incubation of tsHl cells a t 39.5 "C it was of interest to determine whether thesechanges are associated with increased phosphorylation of the a subunit of eIF2. We have analyzed the stateof phosphorylation of eIF-2 by two-dimensional gel electrophoresis and immunoblotting of wholecell extracts, as described previously forHeLaand other cell types (1, 4, 22, 23). Fig. 3 (A-C) illustrates the results from two separate sets of tsHI cell extracts and one set of wild-type cell extracts, prepared from cells exposed to 34 or 39.5 "C. In two of the experiments, cells were also returned to 34 "C after a period at 39.5 "C. The data show

769

that thereis an increase in phosphorylation of eIF-Z(a) at the nonpermissive temperature in tsH, cells which, although small, is reproducible. This change in eIF-2 phosphorylation is not observed in wild-type cells exposed to 39.5 "C.The data in Fig. 3 (A-C) have been quantified by laser densitometry and are presented in Table 11. Statistical analysis of these figures (plus two additional sets not shown) indicates that in tsH, cells the proportion of phosphorylated eIF-Z(a) rises from 5.5 & 2.4% to 17.2 & 3.9% (mean & S. E.) after the temperature shift. This difference is significant at a probability of p < 0.05. The results of the temperature reversal experiment (Fig. 3B) indicate that the level of phosphorylation at 39.5 "C of approximately 16% (which was associated with a 66% inhibition of protein synthesis) was lowered to less than 1%upon restoration of the permissive temperature. Under these conditions protein synthesis recovered completely (data not shown). Because of the limited number of our analyses, it is not possible to say whether there are any real differences in extent of phosphorylation between tsH, cells in suspension and monolayer, but the phosphorylation state of eIF-Z(a) in cells growing in either mode responds in a qualitatively similar manner to thetemperature shift (data not shown). Analyses of the same whole cell lysates for possible covalent modifications of initiation factors eIF-2P (Fig. 3,A-C) or eIF-4B (Fig. 30) failed to reveal any consistent differences between the permissive and nonpermissive temperatures. DISCUSSION

Previous work has established a strongcorrelation between specific temperature-sensitive mutations in aminoacyl-tRNA synthetases and inhibition of polypeptide chain initiation in CHO cells incubated at nonpermissive temperatures (9, 16). The data presented here indicate that the large decrease in the rate of polypeptide chain initiation which occurs when tsHl cells are exposed to 39.5 "C is due to a decline in the activity of the guanine nucleotide exchange factor, GEF, responsible for the recycling of initiation factor eIF-2 between successive rounds of protein synthesis. This is accompanied by a small but significant increase in the proportion of eIF-2 which is phosphorylated on its a subunit. A model for the stoichiometric sequestration and inactivation of limiting amounts of GEF by eIF-2a(P) in reticulocytes has been proposed which explains how partial phosphorylation of eIF-2 can lead to an almost total shutdown of protein synthesis (24). In the present system, an increase of only about 12% in the extent of eIF-P(a) phosphorylation is observed in cells which exhibit a 60-80% decrease in protein synthesis. The majority of the decrease in protein synthesis can be accounted for by impaired GEF (and hence eIF-2) activity. If the sequestration model i s applicable to this system, therefore, it would implya GEF:eIF-2 molar ratio of around 1:7. However, it isimportant to stress that thereis no direct evidence either for or against formation of stable, inactive complexes between eIF-Sa(P) and GEFin tsH, cells at 39.5 "C, and other mechanisms forreversible inactivation of GEF (including phosphorylation of GEF itself) cannot be ruled out. The change in the phosphorylation state of eIF-P(a) on shifting tsH, cells from 34 to 39.5 "C suggests a change in the relative activities of protein kinase(s) and/or phosphatase(s) acting on this initiation factor. However, the effect is likely to be a subtle one since we only observe a small net change in the phosphorylation of eIF-2(a). Previously we have been unable to observe a difference in the abilities of extracts from fed and amino acid-starved cells to phosphorylate eIF-2 in vitro (25, 26), but it is not certain that such an approach is

Control Temperature-sensitive Initiation in of

770

34"

34"

CHO Cells

34" 39.5"

39.5"

39.5"

39.5"

+

39.5" 34"

39.5"

4

34O

PH

t

*

eIF-46

FIG. 3. Analysis of eIF-S(a), eIF-2@),and eJF-4B in extracts from tsHl and wild-type cellsby twodimensional gel electrophoresis and immunoblotting. tsH, cells (A, B, and D)and wild-type (wt)cells ( C ) were incubated as monolayers at 34 "C. Where indicated, cells were rapidly shifted up to 39.5 "C for 45 min and, in some experiments, a fraction of these cells were then returned to 34 "C for a further 45 min. At the appropriate times, the cells were washed three times with cold phosphate-buffered saline by rapidly rinsing the cell monolayers and the cells were immediately lysed in 9.8 M urea, 2% Nonidet P-40, 2% pH 3.5-10 Ampholines, 1% 2mercaptoethanol containing 50 mM NaF and IO mM EDTA, as described previously (22). The whole cell lysates were subjected to isoelectric focusing in the first (horizontal) dimension followed by sodium dodecyl sulfate gel electrophoresis in the second (vertical) dimension; the directions of increasing isoelectric point and relative molecular mass are indicated. The proteins were transferred electrophoretically to nitrocellulose paper and the or with appropriate regions of the blots were incubated with affinity-purified antibodies to eIF-Z(a) and eIF-2(@) an antiserum to eIF-4B (1, 4, 22). The bound antibodies were detected by incubation with '2sI-labeled second antibodies followed by autoradiography. The autoradiograms are shown. A-C, incubation with a mixture of rabbit anti-eIF-B(a) and anti-eIF-2(@)antibodies; D,incubation with goat anti-eIF-4B antibodies. Arrows indicate the positions of the appropriate initiation factors; eIF-2dP) is the phosphorylated form of eIF-Z(a). Note that the relative positions of the eIF-Z(a) and eIF-2(@) spots vary between gels due to slight differences in pH gradients in the first dimension.

TABLE I1 Phosphorylation status of eIF-2a in tsH1 and wild-type cellsat 34 and 39.5 "C Cells were treated as described in Fig. 3. The extent of phosphorylation of eIF-2n was determined and quantified by scanning the autoradiograms in two dimensions with a laser densitometer (LKB). Experiments 1-111 correspond to wnek A-C in Fig. 3. Experiment

type

I

HI

I1

HI

111

Wild-type

Temperature Phosphorylation treatment

of eIF-2u

"C

%

34 39.5 34 39.5 39.5 + 34 34 39.5 39.5 + 34

2.1 22.6 1.0 15.5 0.5 3.4 5.6 7.5

sufficiently sensitive to detect very small changes in protein kinase activity. Wehave observed' thateIF-2(a)protein phosphatase activity isvery low in tsH, cells, suggesting that minorchangesinkinaseactivity,exerting a n effectover relatively prolonged periods of time, could lead to an altered steady state of phosphorylation of the factor. Alternatively, a change in the availability of eIF-2 as a substrate for kinase(s) or phosphatase(s) at the nonpermissive temperature could A. Galpine and M. J. Clemens, unpublished results.

result in a change in its phosphorylation state without any modulation of kinase or phosphatase levels themselves. Detailed analysesof the kineticsof phosphorylation/dephosphorylation and of the subcellular distribution of eIF-2 and its possible association with other proteins of the translational machinery a t 34 and 39.5 "C are required to test the feasibility of these ideas. It is of interest that the additionof eIF-2 or GEFto a cellfree system from tsH, cells exposed to 39.5 "C not only restores protein synthetic activity (Fig. 2) but also renders the system responsive to the stimulatoryeffects of the cap-binding initiation factorcomplex, eIF-4F (TableI). This suggests that, not surprisingly, cap-dependent mRNA binding to ribosomes only becomes rate-limiting in the presence of excess 40 S initiation complexes generated by exogenous or endogenous eIF-2. Unlike the situation in cell-free systems from Ehrlich ascitescells inhibited by heat shock (2), eIF-4F alone had no effect on extracts from tsHl cells subjected to the nonpermissive temperature. We have not assayed the activity of individual mRNA binding factors in tsHlcell extracts but could detect no change in distributionof the multiple forms of eIF-4B ontwo-dimensional gels (Fig. 3). Again, this distinguishes the response from that due toa 45 "C heat shock (1) and suggests that the control mechanisms in these two systems are different. The data presented heresuggest a possible mechanism for the inhibition of initiation at thenonpermissive temperature

Control of Temperature-sensitive Initiation in in CHO cells with temperature-sensitive aminoacyl-tRNA synthetases. Nevertheless, the question still remains of how a number of independent mutations in different synthetase genes, observed in tsH, (16), Leu-21 (27), and Arg-l(28)cells, all exert a rapidregulatory influence on initiation at thelevel of eIF-2 activity (9). It may be that the rate of polypeptide chain initiation is normally coupled to aminoacyl-tRNA synthetase activity and the rate of chain elongation, both of which are known to be depressed at the nonpermissive temperature (16, 27, 28). This coupling could occur by a mechanism involving the phosphorylation of eIF-2. The elucidation of this regulatory pathway is likely to be important for a fuller understanding of the control of mammalian protein synthesis. Acknowledgments-We thank Drs. Chris Proud and Bill Merrick for gifts of rat liver eIF-2 and reticulocyte eIF-4F, respectively. Excellent technical help was provided by Vivienne Tilleray and Eileen Stewart. We thank Vivienne Marvel1 and Barbara Bashford for preparation of the manuscript.

REFERENCES 1. Duncan, R., and Hershey, J. W. B. (1984) J. Biol. Chem. 2 5 9 , 11882-11889 2. Panniers, R., Stewart, E. B., Merrick, W. C., and Henshaw, E. C. (1985) J. Biol. Chem. 260,9648-9653 3. Rudland, P. S., Weil, S., and Hunter, A. R. (1975) J. Mol. Biol. 96,745-766 4. Duncan, R.,and Hershey, J. W. B. (1985) J. Biol. Chem. 260, 5493-5497 5. van Venrooij, W. J. W., Henshaw, E. C., and Hirsch, C. A. (1972) Bwchim. Biophys. Acta 2 5 9 , 127-137 6. Pain, V. M., and Henshaw, E. C. (1975) Eur. J. Biochem. 57, 335-342 7. Pain, V. M., Lewis, J. A., Huvos, P., Henshaw, E. C., and Clemens, M. J. (1980) J. Biol. Chem. 255,1486-1491 8. Austin, S. A., and Clemens, M. J. (1981) Biosci. Rep. 1 , 35-44 9. Austin, S. A., Pollard, J. W., Jagus, R., and Clemens, M. J, (1986) Eur. J. Biochem. 157,39-47

CHO Cells

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10. Farrell, P. J., Hunt, T., and Jackson, R. J. (1978) Eur. J. Biochem. 89,517-521 11. Leroux, A., and London, I. M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,2147-2151 12. Clemens, M. J., Pain, V.M., Wong, S.-T., and Henshaw, E. C. (1982) Nature 296.93-95 13. Siekierka, J., Mauser, L., and Ochoa, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,2537-2540 14. Pain, V.M., and Clemens, M. J. (1983) Biochemistry 2 2 , 726733 15. Panniers, R., and Henshaw, E. C. (1983) J. Biol. Chem. 2 5 8 , 7928-7934 16. Thompson, L.H., Harkins, J. L., and Stanners,C. P. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 3094-3098 17. Pollard, J. W. (1984) in A Laboratory Manwl of Methods in Molecular Biology (Walker, J. M., ed) Part 1, pp. 75-80, Humana Press, Clifton, NJ 18. Stanners, C. P., Elicieri, G. L., and Green, H. (1971) Nature New Biol. 230,52-54 19. Matts, R. L., and London, I. M.(1984) J. Bwl. Chem. 259,67086711 20. Proud, C. G., Clemens, M. J., and Pain,V. M. (1982) FEBS Lett. 148,214-220 21. Grifo, J. A., Tahara, S. M., Morgan, M. A., Shatkin, A. J., and Merrick, W. C. (1983) J. Biol. Chem. 2 5 8 , 5804-5810 22. Duncan, R., and Hershey, J. W. B. (1983) J. Biol. Chem. 2 6 8 , 7228-7235 23. Samuel, C. E., Duncan, R., Knutson, G. S., and Hershey, J. W. B. (1984) J. Biol. Chem. 259,13451-13457 24. Siekierka, J., Manne, V., and Ochoa, S. (1984) Proc. Natl. Acad. Sci. U. S. A. 8 1 , 352-356 25. Austin, S. A., and Clemens, M. J. (1981) Eur. J. Biochem. 117, 601-607 26. Clemens, M. J., Austin, S. A., Kruppa, J., Proud, C. G., and Pain, V. M. (1984) in Protein Synthesis-Translational and Posttranslational Events (Abraham, A. K., Eikhom, T. s., and Pryme, I. F., eds) pp. 389-408, Humana Press, Clifton, NJ 27. Thompson, L. H., Stanners, C. P., and Siminovitch, L. (1975) Somatic Cell Genet. 1,187-208 28. Thompson, L. H., Lofgren, P. J., and Adair, G.M. (1977) Cell 1 1 , 157-168